CN101353648A - Method for extracting hybrid rumex superoxide dismutase - Google Patents
Method for extracting hybrid rumex superoxide dismutase Download PDFInfo
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- CN101353648A CN101353648A CNA2007101193618A CN200710119361A CN101353648A CN 101353648 A CN101353648 A CN 101353648A CN A2007101193618 A CNA2007101193618 A CN A2007101193618A CN 200710119361 A CN200710119361 A CN 200710119361A CN 101353648 A CN101353648 A CN 101353648A
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- rumex acetosa
- hybrid rumex
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Abstract
The invention discloses an extraction method of a hybrid sour dock SOD; the extraction method of the hybrid sour dock SOD provided by the invention comprises the following steps: the hybrid sour dock is pre-processed to obtain crude extract liquid, an ultrafiltration membrane with 60,000 dalton of intercept molecular weight is used for carrying out ultrafiltration process to the crude extract liquid to obtain ultrafiltrate, and an ultrafiltration membrane with 10,000 dalton of intercept molecular weight is used for carrying out concentration to the crude extract liquid to obtain concentrated liquid; the preparation of the crude extract liquid by pre-processing the hybrid sour dock comprises the following steps: the hybrid sour dock is smashed by a waring blender, the smashed tissue is centrifugated by slurry, precipitation and residue are removed and supernatant is retained as the crude extract liquid; the invention introduces ultrafiltration technology, applies two ultrafiltration membranes with different intercept molecular weight to separate and collect the crude extract liquid, can save ammonium sulfate, has no phase change in the extraction process, is beneficial to keeping the enzyme activity of SOD, does not need to go through the process of column chromatographic separation and has the advantages of simple operation process and no environment pollution, etc.
Description
Technical field
The present invention relates to extract the method for plant superoxide-dismutase (SOD), particularly relate to a kind of method of extracting hybrid rumex acetosa SOD.
Background technology
Superoxide-dismutase (SOD) is one of biological important protective enzyme, and energy enhancing body anti-adversity ability is anti-aging.Hybrid rumex acetosa is to adopt the wild garden sorrel in different areas to cultivate the perennial species that form by genetically engineered.It has biological characteristicses such as high-quality, high yield, speed are livings, cold-resistant, drought-enduring, salt tolerant alkali, is suitable for salt marsh, dust storm, desertification soil and grows, and in China most of area of the South and the North plantation is arranged all.The cauline leaf of hybrid rumex acetosa up to more than 30%, is only second to soybean kernel at the contained leaf protein of stooling stage, also contains rich in amino acid, VITAMIN and trace element.Through measuring, contain higher SOD in the hybrid rumex acetosa.
Existing hybrid rumex acetosa SOD extractive technique generally is that hybrid rumex acetosa is handled the centrifugal crude extract that obtains in back, through steps such as 25-35% ammonium sulfate precipitation, 85-95% ammonium sulfate precipitation, Sephadex G-25 desalination and Sephadex G-100 column chromatographies, can obtain the thick enzyme of SOD.
Summary of the invention
The purpose of this invention is to provide a kind of method of extracting hybrid rumex acetosa SOD simply and easily.
The method of extraction hybrid rumex acetosa SOD provided by the present invention, be to be that 60,000 daltonian ultra-filtration membrane uf processing hybrid rumex acetosa crude extracts obtain ultrafiltrated with molecular weight cut-off, be 10,000 daltonian ultra-filtration membrane ultrafiltration concentration liquid with molecular weight cut-off again, the concentrated solution dehydration obtains hybrid rumex acetosa SOD; Described hybrid rumex acetosa crude extract be with hybrid rumex acetosa smash to pieces, the centrifugal supernatant liquor that obtains of homogenate.
Described with hybrid rumex acetosa smash to pieces, homogenate is centrifugal is that hybrid rumex acetosa is smashed to pieces with tissue mashing machine, smashs tissue to pieces after homogenate, the centrifugal condition is 8000/ minute.
Aborning, generally making the mode of concentrated solution dehydration is lyophilize.
In the present invention, using molecular weight cut-off is 60,000 daltonian ultra-filtration membrane uf processing crude extracts, and operating pressure is 0.15MPa during ultrafiltration, obtains ultrafiltrated, and the molecular weight cut-off of ultra-filtration membrane is selected 60,000 dalton.The kit form of ultra-filtration membrane can have various forms, and commonly used have a hollow-fibre membrane.
When using molecular weight cut-off and be 10,000 daltonian ultra-filtration membranes ultrafiltrated being handled, operating pressure is 0.15MPa, is concentrated.The general selection of the molecular weight cut-off of liquid ultra-filtration membrane 10,000 dalton, its kit form can be hollow-fibre membrane.
The technical process of extraction hybrid rumex acetosa SOD as shown in Figure 1, the present invention adopts ultra-filtration technique to separate, concentrate hybrid rumex acetosa SOD crude extract dexterously, not only can save the existing used ammonium sulfate in the hybrid rumex acetosa SOD technology that extracts, and leaching process does not have phase transformation, help keeping alive the stablizing of SOD enzyme, the product enzymic activity that obtains is higher.Simultaneously technology of the present invention need not passed through the column chromatography for separation process, and it is simple to have an operating process, advantages such as environmentally safe.
Description of drawings
Fig. 1 is an extraction process schema of the present invention.
Embodiment
The enzyme activity determination of embodiment 1, SOD
The enzyme activity determination method of SOD adopts the NBT photochemical method, and is specific as follows:
(1) equipment: 752 type spectrophotometers; Desk centrifuge; Illumination box; Refrigerator; 10ml small beaker etc.
(2) reagent: chlorination nitrate blue tetrazolium (NBT), the Shanghai preparation factory that advances
L-methionine(Met) (L-Met), Kangda Amino-acid Factory of Shanghai
Ethylenediamine tetraacetic acid (EDTA) (EDTA), the Beijing Chemical Plant
Riboflavin (VB
2), chemical plant in the west, Beijing
(3) preparation of reaction solution
The preparation of NBT reaction solution: preparation pH7.8, the phosphoric acid buffer 1000ml of 50mmol/L, the L-Met that adds 1.933mg, after treating to dissolve fully, put into 45.8mg NBT, 0.47mg riboflavin (be made into 0.47mg/ml solution, draw 1ml and add) and 29.0mgEDTA, solution final concentration: Met is 13mmol/L; NBT is 6.3 * 10
-3Mmol/L; Riboflavin is 1.3 * 10
-3Mmol/L; EDTA is 0.1mmol/L, places brown reagent bottle, with the shading of black cloth cover, can preserve 1 month in 4 ℃ of refrigerators.
(4) pre-treatment of material
The bright body and function mortar of hybrid rumex acetosa grinds, and according to hybrid rumex acetosa: the ratio of 50mM phosphoric acid buffer=1: 2 adds damping fluid, and centrifugal 10 minutes of 10000rpm gets supernatant liquor and carries out the SOD enzyme activity determination.
(5) measuring method
Draw 30 μ l samples, put into 10ml transparent glass small beaker, add the 3mlNBT reaction solution, mixing is in 28 ℃ of SOD photochmeical reaction chambers, 2 * 15W fluorescent lamp illumination 20 minutes (illumination is 4000 Luxs), measure absorbancy at the 560nm place, make blank, establish 3 repetitions for every group with the NBT reaction solution, whole mensuration process is all carried out under lucifuge or low light condition except that photochmeical reaction.Contrast as test with the standard enzyme of the U.S. " Sigma " company, Bao'an, Shanghai company and the SOD oral liquid of Laolaifu Pharmaceutical Co., Guizhou's production in the test process simultaneously.
(6) calculate
In the formula: CK---the blank transparence
N---diluted sample multiple
A---sample transparence
Enzyme unit: IU/g embodiment 1 alive, extraction hybrid rumex acetosa SOD
Embodiment 2, extraction hybrid rumex acetosa SOD
Get root, stem and the leaf 100kg of fresh hybrid rumex acetosa, smash to pieces with tissue mashing machine, homogenate, 8000rpm frozen centrifugation 10min removes impurity and gets supernatant liquor 70L (crude extract), and enzyme is lived and is 180IU/ml.Is 60,000 daltonian hollow fiber ultrafiltration membrane with crude extract by molecular weight cut-off.Mould material; Polysulfones, membrane area: 0.12m
2, diameter: 10cm, height: 100cm, inner pressed.Crude extract obtains ultrafiltrated 63L through 0.15MPa ultrafiltration 50 minutes, enzyme 173IU/ml alive; Then, be 10,000 daltonian hollow fiber ultrafiltration membrane with ultrafiltrated by molecular weight cut-off, mould material: polysulfones.Membrane area: 0.12m
2, diameter: 10cm, height: 100cm, inner pressed, ultrafiltrated obtains concentrated solution 3L through 0.15MPa ultrafiltration 1 hour, enzyme 3584IU/ml alive.The concentrated solution lyophilize obtains 360 gram dry powder, enzyme 28186IU/g SOD alive, the rate of recovery about 80.2%.
Embodiment 3, extraction hybrid rumex acetosa SOD
Get root, stem and the leaf 100kg of fresh hybrid rumex acetosa, smash to pieces with tissue mashing machine, homogenate, 8000rpm frozen centrifugation 10min removes impurity and gets supernatant liquor 70L (crude extract), and enzyme is lived and is 180IU/ml.Is 60,000 daltonian flat plate ultrafiltration membranes with crude extract by molecular weight cut-off.Mould material: polysulfones, membrane area: 0.15m
2, long: 30cm, wide: 15cm, height: 5cm.Crude extract obtains ultrafiltrated 67L through 0.17MPa ultrafiltration 45 minutes, and enzyme is lived and is 163IU/ml; Then, be 10,000 daltonian flat plate ultrafiltration membranes with ultrafiltrated by molecular weight cut-off.Mould material: polysulfones, membrane area: 0.15m
2Long: 30cm, wide: 15cm, height: 5cm.Ultrafiltrated obtains concentrated solution 2.8L through 0.17MPa ultrafiltration 50 minutes, and enzyme is lived and is 3682IU/ml.The concentrated solution lyophilize obtains 330 gram SOD dry powder, and enzyme is lived and is 32253IU/g, the rate of recovery about 84.5%.
Embodiment 4, extraction hybrid rumex acetosa SOD
Get root, stem and the leaf 100kg of fresh hybrid rumex acetosa, smash to pieces with tissue mashing machine, homogenate, 6000rpm frozen centrifugation 10min removes impurity and gets supernatant liquor 70L (crude extract), and enzyme is lived and is 180IU/ml.Is 60,000 daltonian rolling ultra-filtration membranes with crude extract by molecular weight cut-off.Mould material: polyacrylonitrile, membrane area: lateral area: 3200cm
2, cross-sectional area: 2.4cm
2Crude extract obtains ultrafiltrated 66L through 0.17MPa ultrafiltration 48 minutes, and enzyme is lived and is 162IU/ml; Then, be 10,000 daltonian rolling ultra-filtration membranes with molecular weight cut-off.Mould material: polyacrylonitrile, membrane area: lateral area: 3200cm
2, cross-sectional area: 2.4cm
2, ultrafiltrated obtains concentrated solution 2.5L through 0.17MPa ultrafiltration 55 minutes, and enzyme is lived and is 4124IU/ml.The concentrated solution lyophilize obtains 310 gram SOD dry powder, and enzyme is lived and is 34874IU/g, the rate of recovery about 85.8%.
Claims (5)
1, a kind of method of extracting hybrid rumex acetosa SOD, be to be that 60,000 daltonian ultra-filtration membrane uf processing hybrid rumex acetosa crude extracts obtain ultrafiltrated with molecular weight cut-off, be that 10,000 daltonian ultra-filtration membrane ultrafiltration concentration liquid obtain concentrated solution with molecular weight cut-off again, the concentrated solution dehydration obtains hybrid rumex acetosa SOD; Described hybrid rumex acetosa crude extract be with hybrid rumex acetosa smash to pieces, the centrifugal supernatant liquor that obtains of homogenate.
2, the method for extraction hybrid rumex acetosa SOD according to claim 1 is characterized in that: described with hybrid rumex acetosa smash to pieces, homogenate is centrifugal is that hybrid rumex acetosa is smashed to pieces with tissue mashing machine, smashs tissue to pieces after homogenate, the centrifugal condition is 8000 rev/mins.
3, the method for extraction hybrid rumex acetosa SOD according to claim 1 and 2 is characterized in that: the mode of described concentrated solution dehydration is lyophilize.
4, the method for extraction hybrid rumex acetosa SOD according to claim 1 and 2 is characterized in that: described holding back
Molecular weight is that the kit form of 60,000 daltonian ultra-filtration membranes is hollow-fibre membrane, flat sheet membrane or rolled film.
5, the method for extraction hybrid rumex acetosa SOD according to claim 1 and 2 is characterized in that: described molecular weight cut-off is that the kit form of 10,000 daltonian ultra-filtration membranes is hollow-fibre membrane, flat sheet membrane or rolled film.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101193618A CN101353648A (en) | 2007-07-23 | 2007-07-23 | Method for extracting hybrid rumex superoxide dismutase |
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|---|---|---|---|
| CNA2007101193618A CN101353648A (en) | 2007-07-23 | 2007-07-23 | Method for extracting hybrid rumex superoxide dismutase |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955920A (en) * | 2010-10-13 | 2011-01-26 | 中国石油大学(华东) | Separation technology for gene recombinant heat-resistant manganese superoxide dismutase |
| CN102586200A (en) * | 2011-01-12 | 2012-07-18 | 龚森淼 | Process method for extracting superoxide dismutase from root, stem and leaf of burdock |
| CN104450635A (en) * | 2014-12-12 | 2015-03-25 | 邢台怡通生物科技有限公司 | Method for extracting and purifying superoxide dismutase (SOD) from saccharomyces cerevisiae |
-
2007
- 2007-07-23 CN CNA2007101193618A patent/CN101353648A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955920A (en) * | 2010-10-13 | 2011-01-26 | 中国石油大学(华东) | Separation technology for gene recombinant heat-resistant manganese superoxide dismutase |
| CN102586200A (en) * | 2011-01-12 | 2012-07-18 | 龚森淼 | Process method for extracting superoxide dismutase from root, stem and leaf of burdock |
| CN102586200B (en) * | 2011-01-12 | 2015-06-10 | 龚森淼 | Process method for extracting superoxide dismutase from root, stem and leaf of burdock |
| CN104450635A (en) * | 2014-12-12 | 2015-03-25 | 邢台怡通生物科技有限公司 | Method for extracting and purifying superoxide dismutase (SOD) from saccharomyces cerevisiae |
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Open date: 20090128 |